Mewara et al. Parasites & Vectors (2018) 11:281 https://doi.org/10.1186/s13071-018-2854-0

RESEARCH Open Access Rapid identification of medically important mosquitoes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Abhishek Mewara1*, Megha Sharma1, Taruna Kaura1, Kamran Zaman1, Rakesh Yadav2 and Rakesh Sehgal1

Abstract Background: Accurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it. Methods: Mosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens. Results: Reproducible MALDI-TOF MS spectra spanning over 2–14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55–60 for Anopheles,80–100 for Aedes,30–60 for Culex and 45–50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8). Conclusions: MALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs. Keywords: MALDI-TOF MS, Mosquitoes, Anopheles, Aedes, Culex, Armigerus, ITS2 PCR, Rapid identification

* Correspondence: [email protected] 1Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mewara et al. Parasites & Vectors (2018) 11:281 Page 2 of 9

Background mosquitoes collected from Europe and added ten new Mosquitoes are the most important dipteran vectors species [20]. Few authors have also made use of cluster implicated in about 90% of all vector-borne diseases analysis to depict differences among the species of mos- (VBD) infecting mankind [1]. Nearly one million people quitoes tested [8, 21]. MALDI-TOF MS based identifica- succumb to mosquito-transmitted diseases (MTD) every tion relies on the protein signatures of tested species year worldwide [2]. While several endemic countries and may show variations in response to environmental continue to struggle with the rampant malaria, filariasis and other factors [14, 15, 22]. It is, therefore, pertinent and dengue, emergence and re-emergence of other to evaluate the MALDI-TOF MS protein profiles from VBDs in relatively naive geographical areas pose con- different geographical regions. The current study thus tinuous threat to health services. A glaring example is aimed to harness this technique for protein profiling of that of chikungunya which has spread over the last 15 the commonly encountered mosquito species in North years with outbreaks reported across the globe [3]. Inter- India with the objective of creating a database and evalu- estingly, a better adaptability of the virus to the switched ating the robustness of the database by blinded-testing over vector i.e. from Aedes aegypti to Aedes albopictus, of mosquito specimens. contributed significantly to the massive spread of the disease [4]. Similarly, ongoing transmission of several Methods other MTDs such as Zika virus disease, West Nile fever Mosquito larvae collection, maintenance and and yellow fever highlight the importance of studying morphological identification the various factors associated with the related vectors The aquatic breeding habitats of mosquitoes were surveyed [5, 6], to enable a better understanding of the trans- from various rural, urban and slum areas of Chandigarh, a mission dynamics and institution of effective control Union Territory in North India (30°73'33"N, 76°77'94"E), measures. In the absence of a specific therapy or vac- from March to October 2016. According to the size of a cines against most of the pathogens, vector control site, a representative number of dips were taken for collec- remains the mainstay for controlling the MTDs, tion using standard dipper (350 ml, minimum of 6 and where entomological surveys play a vital role. maximum of 30 dips) from each type of water body from The morphological identification of mosquitoes for edges of sites, around vegetation, and shallow areas. Mos- delineation of genus and species based on specific taxo- quito larvae of four genera, Anopheles, Aedes, Culex and nomical keys has long been the basis of entomological Armigerus, were found to be prevalent. The anopheline surveys. However, reliability of such identification breeding was found mostly in water collections and fields remains questionable owing to loss of body structures around cattle sheds, and edges of permanent rivers from during collection, transport and storage and more August to October; Culex spp. were found mostly in parks, importantly due to presence of sibling-species and inter- near vegetation, around cattle sheds and in ditches from species within the same species complex [7]. Molecular May to October; Armigeres spp. were found more in dense methods, mainly targeting the nuclear internal tran- vegetation with long persisting water logging from July to scribed spacer 2 region (ITS2) and other gene targets for September; and Aedes spp. were found in water collections mosquito genera such as Anopheles, Aedes and Culex in parks, small streams, coolers, artificial containers and are the current gold standard for correct identification pots from March to September. The larvae were identified [8]. Although the molecular techniques are very sensitive morphologically and separated in plastic trays on the basis and specific, DNA extraction from mosquitoes is a of their genera. They were fed on a protein rich diet con- laborious process requiring technical expertise and dedi- sisting of a mixture of finely powdered yeast extract and cated personnel. Matrix-assisted laser desorption/ dog biscuits in the ratio of 3:2 and were reared up to the ionization time-of-flight mass spectrometry (MALDI- adult stage. All the adult mosquitoes were morphologically TOF MS), with its unique ability of protein profiling, identified under the stereomicroscope (AO SteroStar, may provide an alternative to the morphological and American Optical Corporation, New York, USA) using molecular characterization of insects. After proving its standard morphological keys [23], labeled and kept in sep- worth in reliable identification of bacteria, fungi, giant arate Eppendorf tubes until further analysis. viruses and Archea [9], the technique has been success- fully employed for arthropods like Drosophila [10], ITS2 polymerase chain reaction (PCR) aphids [11], Culicoides species [12], ticks [13], sand flies The DNA was extracted from legs of the adult mosquitoes [14], tsetse flies [15], fleas [16] and also mosquitoes [8, using tissue DNA extraction kits as per the manufacturer’s 17–19]. Yssouf et al. [19] carried out MALDI-TOF MS instructions (Qiagen India Pvt. Ltd., New Delhi, India). based identification of 20 mosquito species prevalent in The extracted DNA was amplified by PCR as previously Senegal, created a database for them, and later on evalu- described using specific primers targeting ITS2 region ated the robustness of the database by testing [24] (forward: 5'-TGT GAA CTG CAG GAC ACA T-3' Mewara et al. Parasites & Vectors (2018) 11:281 Page 3 of 9

and reverse: 5'-TAT GCT TAA ATT CAG GGG GT-3'). Parameters for MALDI-TOF MS analysis All the reagents used for PCR were obtained from Using the linear positive ion mode with a mass/charge Sigma-Aldrich (Sigma-Aldrich Chemicals Pvt. Ltd., (m/z) range of 2–20 kDa and the Flex Control software Bangalore, India). The 50 μl reaction mixture contained (Bruker Daltoniks), each well was subjected to 300 laser 10 mM each of the forward and reverse primers, 100 shots aimed at six different regions of the well with laser mM each of the dNTPs, 2 U of Taq DNA polymerase firing set at 50 shots/fire. All specimens belonging to the and 2 μl of the extracted DNA. The amplification was same species produced spectra that spanned over the 2– performed in a thermocycler (Thermo Fisher Scientific, 14 kDa m/z range with comparable peak patterns, inten- Vantaa, Finland) using the following parameters: initial sity of peaks and the overall quality of spectra. denaturation at 94 °C for 5 min, 36 cycles each of de- naturation at 94 °C for 1 min, annealing at 59 °C for 1 Spectra processing and database creation min, elongation at 72 °C for 1 min, followed by final Five morphologically and ITS2 PCR-confirmed female extension at 72 °C for 5 min. PCR grade water was used mosquitoes of each species were processed for creation as negative control in each reaction. The PCR products of the database. The spectra were processed using the were analyzed by 1.2% agarose gel electrophoresis con- Flex Analysis software for peak smoothening and base- taining ethidium bromide and visualized in ultra-violet line subtraction. The spectra generated from four spots light in a gel documentation system (AlphaImagerTM of the same specimen were compared using the ClinPro- EC, Protein-Simple, San Jose, CA, USA) to detect spe- Tools v.2.2 software (Bruker Daltoniks) to check for cific bands for different genera: 500 bp for Anopheles reproducibility of the spectra. Out of these four spectra, spp.; 300 bp for Aedes spp.; 450 bp for Culex spp.; and the one having maximum number of peaks with > 50% 420 bp for Armigeres spp. intensity was selected. These selected spectra from all The PCR products were sequenced using the Big-Dye five representative specimens were used for creation of Terminator sequencing kit in an ABI 3130 Genetic the database using Biotyper v.3.0. The same was done Analyzer automated sequencer (Applied Biosystems, for all the nine species included in the study. Foster City, CA, USA) as per the manufacturer’sinstruc- tions. The sequencing primers were identical to the PCR Identification of signature peaks for each species primers. The mass lists of all the specimens used for creation of the database, i.e. five female specimens per mosquito MALDI-TOF MS analysis species, were exported to Microsoft Excel sheets for ana- Sample preparation lysis. The peaks with a relative intensity of less than 5% The cephalothorax of the adult mosquitoes was selected for were excluded, and those with a minimum signal-to- MALDI-TOF MS analysis. Briefly, each mosquito was ex- noise threshold of 4.0 and an aggregation of 800 ppm amined under the stereomicroscope and its cephalothorax were selected. The optimal peaks thus obtained from the was carefully removed from the rest of the body (legs, nine mosquito species were then compared to identify wings, abdomen), and transferred to a 1.5 ml Eppendorf genus-specific and species-specific biomarker peaks tube containing 20 μl of 50% formic acid. The cephalo- among them. thorax was manually grinded with formic acid with the help of a fused tip. A solution containing 60% acetonitrile Cluster analysis (ACN) and 0.3% tri-fluoroacetic acid (TFA) obtained from A cluster analysis based on protein profiling was carried Sigma-Aldrich was prepared, and 7.5 μl of it was added to out to study the differences among the tested mosquito separate tubes. To this solution, 5 μl of the mosquito hom- species with an anticipation that the closely related spe- ogenate was added and mixed thoroughly. One microliter cies will tend to cluster together, and away from the of this final solution was spotted onto the 96-well stainless unrelated ones. This was done to check for the robust- steel plate, air dried, and overlaid with 1 μlofHCCAmatrix ness of the database in identifying inter- and intra- (α-cyano-4-hydroxycinnamic acid) prepared by dissolving species variations. One spectrum of each species was the matrix powder into a solution containing 50% ACN, 47. randomly chosen to create MSP (mean spectrum projec- 5% molecular-grade water and 2.5% TFA. Once the plate tion) for each of the nine mosquito species. Using Bioty- was completely dry, it was inserted into the port of Micro- per v.3.0, a dendrogram was constructed based on the flex MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, principal components analysis (PCA). Germany) to take the reading. Each specimen was spotted onto four different wells to check reproducibility. A blank Blinded-testing of coded mosquito specimens tube with no mosquito parts in it underwent all the pro- Sixty fresh specimens collected from the same sites and cessing steps each time. E. coli ATCC 25922 was also spot- confirmed by morphological and molecular identifica- tedalongwitheachplatetorunasacontrol. tion by ITS2 PCR were coded and subjected to blinded- Mewara et al. Parasites & Vectors (2018) 11:281 Page 4 of 9

testing. Of the nine species, seven specimens of each 30–60 for Culex and 45–50 for Armigeres species species (except for Ar. subalbatus for which four speci- (Fig. 1, Additional file 1:FiguresS1-S9).Theoptimal mens were available), were tested by MALDI-TOF MS. peaks selected from among these are represented in The protein extracted from their cephalothorax was Table 1. A few genus-specific signature peaks present spotted onto the 96-well stainless steel plate in tripli- in all species of the same genus were identified. For cates and processed under Flex Control software. The example, peak (m/z) 5243 was present in all anophel- acquired spectra were matched against the created data- ine species, 9310 in both aedine species, and 8167 in base using Biotyper v.3.0. As described previously by all culicine species. Among the three species of Yssouf et al. [20] for European mosquito species, a score Anopheles, peaks 6298 and 8915 were present only in of > 1.8 was used to denote reliable genus and species An. stephensi; 3192, 4428, 6383, 8858 and 10144 only identification, > 1.6 and < 1.79 to denote reliable genus in An. culicifacies; and 8924 only in An. annularis; identification, and a score of < 1.59 was considered while peak 4480 was present in both An. stephensi unreliable. and An. annularis,butnotinAn. culicifacies.Among the two species of Aedes, wide variation among signa- Results ture peaks was present. Among the three Culex spe- Mosquito larvae collection and identification cies, peak 9028 was specific for Cx. vishnui,4686for A total of 2000 mosquito larvae of the four genera Cx. tritaenorhynchus, and 3210 and 5210 for Cx. were reared in the lab up to the adult stage for iden- quinquefasciatus, while peaks 5198 and 6293 were tification. Out of 1000 Culex, 600 Aedes, 200 present in both Cx. tritaenorhynchus and Cx. vishnui, Armigeres and 200 Anopheles larvae, 70, 59, 51, and but not in Cx. quinquefasciatus. The biomass peaks 49% emerged into adults, respectively. A total of nine 4825, 5138, 6387, 8578 and 10995 were present only species were found: three species of Anopheles (An. in Ar. subalbatus. stephensi, An. culicifacies and An. annularis); two species of Aedes (Ae. aegypti,andAe. albopictus); Cluster analysis three species of Culex (Cx. quinquefasciatus, Cx. vish- The three species of Anopheles (An. stephensi, An. annu- nui and Cx. tritaenorhynchus); and one species of laris and An. culicifacies) clustered together. The two Armigerus (Ar. subalbatus). There was full concord- Culex species (Cx. vishnui and Cx. tritaenorhynchus) ance between morphological identification and ITS2 clustered together while Cx. quinquefasciatus stood out PCR sequencing for the identification of mosquito from the other species. Armigeres clustered closer to Ae- species. des than any other species (Fig. 2).

MALDI-TOF MS analysis Analysis of blinded-testing of coded mosquitoes Analysis of signature peaks for different mosquitoes Of the 60 coded mosquito specimens, 58 (96.67%) The average numbers of peaks produced by five fe- were correctly identified with the database with a male specimens of the different mosquito species score of > 1.8, while 2 specimens (both confirmed as were 55–60 peaks for Anopheles,80–100 for Aedes, Cx. quinquefasciatus by ITS2 PCR) were misidentified

Fig. 1 MALDI-TOF MS spectra obtained from the cephalothorax part of nine mosquito species Mewara et al. Parasites & Vectors (2018) 11:281 Page 5 of 9

Table 1 Representative biomarker peaks for nine mosquito species as obtained by MALDI-TOF MS analysis Biomass An. st. An. cu. An. an. Ae. ae. Ae. al. Cx. tr. Cx. vi. Cx. qu. Ar. su. 3192 × 3200 × 3210 × 3402 × × × × 4428 × 4480 × × 4675 × 4686 × 4825 × 4831 × 5138 × 5198 ×× 5210 × 5243 × × × 5505 × 5514 × 6293 ×× 6298 × 6324 × × 6383 × 6387 × 6399 × 6515 × 8042 × × 8056 × 8167 ××× 8565 × 8578 × 8858 × 8915 × 8924 × 9028 × 9310 × × 10144 × 10995 × Abbreviations: Ae. ae. Aedes aegypti, Ae. al. Aedes albopictus, An. an. Anopheles annularis, An. cu. Anopheles culicifacies, An. st. Anopheles stephensi, Ar. su. Armigeres subalbatus, Cu. qu. Culex quinquefasciatus, Cu. tr. Culex tritaenorhynchus, Cu. vi. Culex vishnui

as Cx. vishnui. Both these specimens produced an un- Discussion reliable identification with a score of < 1.6, even on re- In this study, we collected four genera of medically peated testing on the same wells in addition to important mosquitoes from North India and used them different wells with the same homogenates. The blank for creating MALDI-TOF MS protein profiles for rapid always produced a score of < 1 and the E. coli ATCC and accurate identification. While for Ae. aegypti, Ae. 25922 gave a reliable score of > 2 every time when albopictus, An. stephensi and Cx. quinquefasciatus,a matched against the in-built database of the Biotyper. database has been recently created [8, 19], the other five Mewara et al. Parasites & Vectors (2018) 11:281 Page 6 of 9

Fig. 2 Dendrogram constructed by Biotyper for nine mosquito species. Numbers 1, 2 and 3 denote labels of three specimens of each species of mosquito. The cephalothorax part of the mosquitoes was taken for analysis. Abbreviations: Ae. ae, Aedes aegypti; Ae. al, Aedes albopictus; An. an, Anopheles annularis; An. cu, Anopheles culicifacies; An. st, Anopheles stephensi; Ar. su, Armigeres subalbatus; Cu. qu, Culex quinquefasciatus; Cu. tr, Culex tritaenorhynchus; Cu. vi, Culex vishnui species, i.e. An. annularis, An. culicifacies, Cx. vishnui, while the abdomen of the adult mosquito is not pre- Cx. tritaenorhynchus and Ar. subalbatus have not yet ferred as it may contain the residual meal of the insect been evaluated by MALDI-TOF MS. The anophelines which may interfere with the protein peaks [11, 21, 29]. are known vectors of malaria, aedine species spread Yssouf et al. [19] have reported good results by using dengue and chikungunya, culicines spread filariasis mosquito legs for protein extraction; however, we pre- (Cx. quinquefasciatus), Japanese encephalitis and West ferred to use the cephalothorax as it adequately yields Nile fever (Cx. vishnui, Cx. tritaenorhynchus), and Ar. the minimum concentration of 0.2 mg/ml of raw protein subalbatus is implicated in filariasis and Japanese required for generation of optimal spectrum by MALDI- encephalitis. These MTDs pose significant public TOF MS [28]. The legs of the mosquito are less likely to health concerns worldwide as well as in India. The be contaminated with extraneous proteins; however, quick and reliable identification of these mosquito some of the legs can easily be lost during collection, species is essential to implement effective vector con- transportation or storage of mosquitoes, thereby trol measures, in addition to having important epi- decreasing the protein content. Furthermore, in the demiological significance [25]. present study, we used the legs of the mosquitoes for The identification using MALDI-TOF MS is based on molecular identification and the cephalothorax of the signature proteins of the organism and different life- same mosquitoes for MALDI-TOF analysis. The turn- cycle stages of mosquitoes will generate different mass around time was less than 30 minutes per specimen and spectra [26, 27]. We collected larvae from the field, the running cost was less than USD 0.05 per specimen, reared them to adult stage, and used the cephalothorax excluding the cost of the machine. for protein extraction. Muller et al. [8] also used ceph- The spectra generated by the nine mosquito species alothorax for identifying An. gambiae mosquitoes; how- were not only visually distinct but also had specific ever, different body structures like legs of mosquitoes signature peaks. Of the species we tested, Steinmann [19], or even different life-cycle stages like larvae [28] et al. [28] have documented the peaks obtained for and eggs [18] of mosquitoes have been previously used, larvae of Ae. aegypti; while some of their peaks Mewara et al. Parasites & Vectors (2018) 11:281 Page 7 of 9

coincided with Ae. aegypti in our study, the difference in [13], although no general consensus exists regarding the other peaks was anticipated due to the different life stage optimal cut-off for vector identification by MALDI-TOF of mosquito analyzed (larva vs adult). Yssouf et al. [19] MS as of now. With a cut-off of 1.8, 96.67% of the coded have obtained peaks from the legs of Ae. aegypti and Cx. specimens were correctly identified by our database. quinquefasciatus and their peaks vary greatly from those Two C. quinquefasciatus scored poorly even on repeated in our study. This could be attributed to the difference testing, the reasons of which need further exploration. in the body part used for analysis in the two studies, as Similar observation has been made earlier where unreli- also described earlier by Karger et al. [22] for ticks. Spec- able identification (score < 1.8) of three mosquito spe- tral differences have also been previously noted when cies (Culiseta longiareolata, Coquillettidia richiardii and different body parts (cephalothorax vs legs) of flies were Ae. caspius) was attributed to lower spectral quality [20]. tested and may be attributed to difference in the protein More prospective studies will help to validate the data- content [15, 21]. The differences could also be due to base and to define optimal cut-offs for reliable vector intra-specific genetic diversity among specimens col- identification. lected from different environments and geographical MALDI-TOF MS is now being heavily relied upon locations, as reported earlier in anopheline species [30], for the identification of bacteria and other microor- Ae. aegypti [31], Ae. albopictus [32]andCx. quinquefas- ganisms; however, its reliable use for identification of ciatus [33], based on molecular gene targets, and it is vectors encompasses many challenges. First, to enable likely that their protein profiles may also exhibit varia- construction of a reliable database as that for other tions. Further studies investigating the role of environ- pathogens, it is important to take into consideration mental and host factors on genetic and phenotypic several parameters affecting protein profiles in a vec- variations among members of the same species can tor: life-cycle stage, body part, gender differences, contribute to our knowledge of such diversity. genetic diversity, environmental and geographical vari- The dendrogram analysis of the nine species clustered ations, extraction protocol, etc. Complex as it them on the basis of their mass spectra similarities. As appears,thismightbethereasonwhytheMALDI- expected, three of the anophelines (An. stephensi, An. TOF machines do not have in-built databases for vec- culicifacies and An. annularis) clustered together. It was tors until now. The Switzerland-based firm, Mabritec observed that the similarity between An. stephensi and AG, has created a database including 70 arthropod An. annularis was more than that with An. culicifacies. species [7]; however, it needs to be meticulously vali- This corroborates with the reported genomic linkage in dated on species collected from across the globe. Sec- which An. stephensi and An. annularis cluster together ondly, as the identification depends upon protein on the basis of ITS2 spacer and cox2 gene while An. profiling, it is deemed necessary that the integrity of culicifacies only shows some linkage with An. annularis protein is maintained during collection, transporta- on the basis of the NOS gene [30]. Both aedine species, tion, storage and processing of the specimen. Finally, as expected on the basis of their genomic similarity [34], with the known and ongoing genetic diversity occur- clustered closely together. Among the culicine species, ring within sibling-species of mosquitoes, a regular Cx. quinquefasciatus aligned away from Cx. vishnui and update of the databases may be necessary. These limi- Cx. tritaenorhynchus; while it was expected owing to a tationsshouldbeovercomewithtimeasmoredatais unique set of peaks produced only by Cx. quinquefascia- accumulated in this field. tus, this finding could also be plausibly explained on the basis of the genetic diversity of Cx. quinquefasciatus Conclusions which forms a separate clade from the rest of the Culex MALDI-TOF MS appears to be a pragmatic, rapid, accur- species [35]. The last species, Ar. subalbatus, a relatively ate and cost-effective tool for the identification of mosqui- less-studied species, clustered along with aedine species toes which may circumvent the redundancies of instead of forming a separate subgroup. Constituting morphological variations as well as the complexities of nearly 7% of all mosquito species in central India, this DNA-based identification tools. However, more informa- species blooms during monsoons with sewage and dirty tion is required before standardized protocols can be cre- water as its major habitat [36]. Although vast geograph- ated for vectors. Its utility can be expanded to include ical variation has been reported [37], the low heterozy- important arenas like characterization of insecticide resist- gosity between Armigeres and Aedes [38] may explain its ance in vectors, detection of carriage of pathogens, and clustering along with the aedine species. also study of dipteran vectors other than mosquitoes. After creating the database, blinded testing of 60 spec- Thus, MALDI-TOF MS has great potential for the study imens was done to analyze the reliability of identification of vectors and it is worthwhile to accrue more information (using 1.8 as the cut-off score). Yssouf et al. [19] have on its use to enhance the clarity of the scope of its applic- used a cut-off of 1.8 for mosquitoes, and 1.7 for ticks ability in the control of vector-borne diseases. Mewara et al. Parasites & Vectors (2018) 11:281 Page 8 of 9

Additional file Received: 1 December 2017 Accepted: 17 April 2018

Additional file 1: Figure S1. MALDI-TOF MS spectra of three representative specimens of An. stephensi. Figure S2. MALDI-TOF MS spectra of three representative specimens of An. culicifacies. Figure S3. References MALDI-TOF MS spectra of three representative specimens of An. 1. Klempner M. Taking a bite out of vector-transmitted infectious diseases. annularis. Figure S4. MALDI-TOF MS spectra of three representative N Engl J Med. 2008;356:2567–9. specimens of Ae. aegypti. Figure S5. MALDI-TOF MS spectra of three 2. Mcgraw EA, Neill SLO. Beyond insecticides: new thinking on an ancient representative specimens of Ae. albopictus. Figure S6. MALDI-TOF MS problem. Nat Rev Microbiol. 2013;11:181–93. spectra of three representative specimens of Cx. tritaenorhynchus. 3. Burt FJ, Chen W, Miner JJ, Lenschow DJ, Merits A, Schnettler E, et al. Figure S7. MALDI-TOFMSspectraofthreerepresentativespecimensof Chikungunya virus: an update on the biology and pathogenesis of this Cx. vishnui. Figure S8. MALDI-TOF MS spectra of three representative emerging pathogen. Lancet Infect Dis. 2017;3099:1–11. specimens of Cx. quinquefasciatus. Figure S9. MALDI-TOF MS spectra of 4. Tsetsarkin KA, Vanlandingham DL, Mcgee CE, Higgs S. A single mutation in three representative specimens of Ar. subalbatus.(PDF2050kb) chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog. 2007;3:e201. 5. Yakob L, Walker T. Zika virus outbreak in the Americas: the need for novel mosquito control metods. Lancet Glob Heal. 2015;4:e148–9. Abbreviations 6. Weaver S, Reisen W. Present and future arboviral threats. Antivir Res. 2011; ACN: Acetonitrile; HCCA: α-cyano-4-hydroxycinnamic acid; ITS: Internal 85:1–36. transcribed spacer; MALDI-TOF MS: Matrix-assisted laser desorption/ionization 7. Yssouf A, Almeras L, Raoult D, Parola P. Emerging tools for identification of time-of-flight mass spectrometry; MTD: Mosquito-transmitted diseases; arthropod vectors. Future Microbiol. 2016;11:549–66. PCA: Principal components analysis; PCR: Polymerase chain reaction; TFA: Tri- fluoroacetic acid; VBD: Vector-borne diseases 8. Muller P, Pflu V, Wittwer M, Ziegler D, Chandre F, Simard F, et al. Identification of cryptic Anopheles mosquito species by molecular protein profiling. PLoS One. 2013;8:e57486. Acknowledgements 9. Giebel R, Worden C, Rust SM, Kleinheinz GT, Robbins M, Sandrin TR. We thank the Department of Medical Microbiology, Postgraduate Institute of Microbial fingerprinting using matrix-assisted laser desorption ionization Medical Education and Research, Chandigarh, India, for kindly providing time-of-flight mass spectrometry (MALDI-TOF MS): applications and access to the Microflex MALDI-TOF MS (Bruker Daltoniks) for this study. We challenges. App Adv Microbiol. 2010;71:149–84. acknowledge Amit Sharma, Laboratory Assistant, Department of Medical 10. Feltens R, Görner R, Kalkhof S, Gröger-arndt H, von Bergen M. Discrimination Parasitology, PGIMER, Chandigarh, for help in field collection of mosquito of different species from the genus Drosophila by intact protein profiling specimens. using matrix-assisted laser desorption ionization mass spectrometry. BMC Evol Biol. 2010;10:1–10. 11. Perera MR, Flores Vargas RD, Jones MGK. Identification of aphid species Funding using protein profiling and matrix-assisted laser desorption/ionization time- The work was funded by the Postgraduate Institute of Medical Education of-flight mass spectrometry. Entomol Exp Appl. 2005;117:243–7. and Research, Chandigarh, India. The funding body had no role in the 12. Kaufmann C, Steinmann IC, Hegglin D, Schaffner F, Mathis A. Spatio- design of the study, specimen collection, analysis and interpretation of data temporal occurrence of Culicoides biting midges in the climatic regions of and in writing the manuscript. Switzerland, along with large scale species identification by MALDI-TOF mass spectrometry. Parasit Vectors. 2012;5:246. 13. Yssouf A, Flaudrops C, Drali R, Kernif T, Socolovschi C, Berenger J, et al. Availability of data and materials Matrix-assisted laser desorption ionization - time of flight mass spectrometry The data generated or analyzed during this study are included in this published for rapid identification of tick vectors. J Clin Microbiol. 2013;51:522–8. article. Representative sequences were submitted to the GenBank database under 14. Dvořák V, Halada P, Hlaváčkova K, Dokianakis E, Antoniou M, Volf P. the accession numbers MH187962-MH187968 and MH198324-MH198325. Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix- assisted laser desorption/ionization time of flight mass spectrometry. Parasit Authors’ contributions Vectors. 2014;7:21. AM, RY and RS designed the study protocol. MS, TK and KZ executed the 15. Hoppenheit A, Murugaiyan J, Bauer B, Steuber S, Clausen PH, Roesler U. laboratory procedures. AM, MS, TK and RY analyzed and interpreted the Identification of tsetse (Glossina spp.) using matrix-assisted laser results. AM, MS, TK and RY wrote the manuscript. All authors read and desorption/ionisation time of flight mass spectrometry. PLoS Negl Trop approved the final manuscript. Dis. 2013;7:e2305. 16. Yssouf A, Socolovschi C, Leulmi H, Kernif T, Bitam I, Audoly G, et al. Identification of flea species using MALDI-TOF/MS. Comp Immunol Ethics approval and consent to participate Microbiol Infect Dis. 2014;37:153–7. The study was approved by the Institute Ethics Committee (Intramural) of 17. Tandina F, Almeras L, Koné AK, Doumbo OK, Raoult D, Parola P. Use of the Postgraduate Institute of Medical, Education and Research, Chandigarh, MALDI-TOF MS and culturomics to identify mosquitoes and their midgut India. microbiota. Parasit Vectors. 2016;9:495. 18. Schaffner F, Kaufmann C, Pflüger V, Mathis A. Rapid protein profiling facilitates surveillance of invasive mosquito species. Parasit Vectors. 2014;7: Competing interests 142. The authors declare that they have no competing interests. 19. Yssouf A, Socolovschi C, Flaudrops C, Ndiath MO, Sougoufara S, Dehecq J, et al. Matrix-assisted laser desorption ionization - time of flight mass spectrometry: an emerging tool for the rapid identification of mosquito Publisher’sNote vectors. PLoS One. 2013;8:e72380. Springer Nature remains neutral with regard to jurisdictional claims in 20. Yssouf A, Parola P, Lindström A, Lilja T, Ambert GL, Bondesson U. published maps and institutional affiliations. Identification of European mosquito species by MALDI-TOF MS. Parasitol Res. 2014;113:2375–8. Author details 21. Halada P, Hlavackova K, Dvořák V, Volf P. Identification of immature stages 1Department of Medical Parasitology, Postgraduate Institute of Medical of phlebotomine sand flies using MALDI-TOF MS and mapping of mass Education and Research, Sector 12, Chandigarh 160012, India. 2Medical spectra during sand fly life cycle. Insect Biochem Mol Biol. 2018;93:47–56. Microbiology, Postgraduate Institute of Medical Education and Research, 22. Karger A, Kampen H, Bettin B, Dautel H, Ziller M, Hoffmann B, et al. Species Sector 12, Chandigarh 160012, India. determination and characterization of developmental stages of ticks by Mewara et al. Parasites & Vectors (2018) 11:281 Page 9 of 9

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